Advances in understanding of the physiological nature of individual lipoprotein types and the effects on human health make it imperative to understand populations of lipoprotein particles and subforms, each of which is the result of, and participates in, specific metabolic processes. Such processes may be good or bad for a particular patient's health, having consequences for therapeutic efforts, including pharmacological therapy, lifestyle changes, diet changes, or other medical intervention.
Lipoproteins are particles in the blood comprising a lipid particle and a variety of apolipoprotein moieties. Lipoproteins span a wide variety of sizes and apolipoprotein content, each species having a unique metabolic pathway and unique relevance to patient health.
Lipoprotein(a) (Lp(a)) levels in a patient are known to correlate strongly with cardiovascular and metabolic health in a patient. It is a goal of existing Lp(a) assays to determine concentration of Lp(a), described as particle number (PN) and generally expressed in nmol/L from a patient's bodily fluid sample, particularly from blood, serum or plasma. The levels of Lp(a) are generally stratified into risk classifications to determine a patient's health status, trends in health status, whether treatment is necessary, and to monitor treatment.
Additionally, the determination of apolipoprotein content in a patient is known to be useful in determining overall health and for guiding treatment. For example, ApoB is known to be a risk factor of atherosclerosis independent of the lipoproteins to which it is linked. Apo(a) is another such protein with individual characteristics having significance for cardiovascular disease. Apo(a) partly comprises the Lp(a) particle and has a variety of subforms. Apo(a) subforms are derived specifically from the different possible repeated domains. It is composed of five domains called kringles (kringle I-V). Kringle IV type 2 is a repeating structure with from 3 to >50 times on a single Apo(a) molecule. The number of repeats thought to exist is variable in literature. However, kringle IV is itself comprises 10 different sequence/structures that can be recognized. Kringle IV type 2 (KIV2) is the one that repeats. The number of KIV2 repeats gives a molar mass of the total protein anywhere from 200 k Da to 800 k Da. The weight of the apo(a) protein translates to variation in the Lp(a) lipoprotein, which is composed of the lipid particle, one apolipoprotein B (apoB) protein and one apo(a) protein.
Although recent improvements in quantifying particle numbers of the various lipoprotein particles, particularly Lp(a), have been made (see, e.g., Marcovina et al., “Effect on the Number of Apolipoprotein(a) Kringle 4 Domains on Immunochemical Measurements of Lipoprotein(a),” Clin. Chem. 41(2): 246-255 (1995); Marcovina et al., “Identification of 34 Apolipoprotein(a) Subforms: Differential Expression of Apolipoprotein(a) Alleles Between American Blacks and Whites,” Biochem Biophys Res Commun 191:1192-6 (1993); Lackner et al., “Molecular Basis of Apolipoprotein(a) Subform Size Heterogeneity as Revealed by Pulsed-Field Gel Electrophoresis,” J Clin Invest 87:2153-61 (1991); Kraft et al., “Apolipoprotein(a) Kringle IV Repeat Number Predicts Risk for Coronary Heart Disease,” Arterioscler Thromb Vasc Biol. 16(6):713-9 (1996)), particle numbers are only one factor in the health effects of Lp(a). It has been determined that the Lp(a) mass is also a factor. In particular, the number of apo(a) KIV2 domains has been found to contribute to an over counting of Lp(a) particles in a sample (see Id.).
Existing assays such as the Denka Seiken Lp(a)-latex assay, based on the Markovina work have been designed to measure the Lp(a) concentration (particle number) via immunological detection to minimize the kringle repeat size problem. This method, however, may be subject to interference by the variable number of apo(a) KIV2 repeats, as it uses one antibody directed to the variable size KIV2 domain.
Improvements are needed to permit efficient and cost-effective identification of Lp(a) subforms. Existing immunological methods are drawn only to the measurement of particle number and have no consideration of the Lp(a) mass. A full characterization would involve at least two assays to generate a complete measurement of Lp(a) measurement: first an apo(a) protein mass assay and second a molecular weight determination of the subform by amino acid analyzer or gradient gel electrophoresis.
This present invention is directed to an efficient single assay that can be used to determine Lp(a) mass and particle number, curing deficiencies in the art.